JP2632063B2 - Color image light receiving element - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a color image light receiving element, and more particularly to a color image light receiving element utilizing the function of a photosensitive chromoprotein. In other words, by using two types of antibodies having different antigen specificities (Bispecific antibodies: BS antibodies), the received color image information can be efficiently converted into electrical signals by precisely controlling the orientation of the photosensitive chromoprotein. It enables conversion and is used for optical color sensors, high-density optical information recording, and the like.

(Conventional technology) In order for an artificial system to fully exert the function of a protein, not only structural problems such as tertiary structure and quaternary structure of the protein but also rules such as hydrophobicity, hydrophilicity, distribution, and orientation are required. It is necessary to reproduce as much as possible the environment that has been placed in the living body, such as a typical aggregate state. Biological membranes are a good example.Membrane proteins are responsible for many functions, such as substance recognition / transport, information transmission, and energy conversion.However, their properties are directional and their distribution and orientation within the membrane are both front and back. Is considered indispensable for the expression of functions. Photochromic proteins represented by rhodopsin are not exceptional and are characterized by absorbing visible light and converting it into chemical work with high efficiency in a vector manner.

Indeed, bacteriorhodopsin is referred to as a proton pump because it actively transports protons in one direction as a result of light absorption. The visual pigments rhodopsin and bacteriorhodopsin are well-known as the photochromic proteins of rhodopsins, and the latter has attracted attention for its use in biodevices such as optical sensors and optical switches because of its excellent stability in vitro. .

As a means for extracting the optical response of bacteriorhodopsin as a physical signal outside a living body, a method using photoelectric conversion is generally used because it is advantageous for application to devices.

A bacteriorhodopsin film (purple film) has a strict distinction between front and back, and in order to make an element using this, a thin film in which molecules are oriented must be produced. Several efforts for such orientation have hitherto been made, for example, reconstitution into liposomes or black membranes (E. Rack
er et al “J. Biol. Chem.” 249 662, 1974, LADrachev et
al. “FEBS” dett 39 43, 1974); Orientation to charged membranes and ion conversion membranes (G. Fisher et. al “J. Memb. Sci.” 16 391 198)
3, K. Singh et.al “B: Ophys. J.” 31 393, 1980); Orientation control by applying an electric field (K. Nagy “Biochem. Biophys. Res. Comm.
un. " 85 383 1978 and G. Varo." Aeta. Biol. Acad. Sci. Ifu.
ng. " 32 301, 1981); Orientation by gas-liquid interface film (T. Fur.
uno et al "Thin Solid Films" 160 145, 1988).

(Invention Problems to be Solved) conventional methods, for example, surface chemical methods of liposome and representatives when present in biological membranes increases what is oriented in reverse as front and back abundance, H ⁺ of As the transport direction, transport from the outside to the inside of the liposome is observed, but it is said that not all bacteriorhodopsin molecules are oriented in the same direction. On the other hand, in an electrostatic orientation method such as application of an electric field, the bacteriorhodopsin-lipid complex (purple membrane) has seven helices per molecule penetrating the membrane, and the sum of the dipole moments caused by them is finite. (About 10 ⁶ teva per piece of purple membrane). Nagy and Varo made such an electrodeposited thin film of bacteriorhodopsin and made a dry cell in which the thin film was sandwiched between two conductive electrode plates to obtain a photovoltaic response. Sora et al. (JP-A-62-63)
823) uses a similar electrodeposition film to build a more sophisticated optical sensor. However, the electrodeposited film produced in this manner has a large thickness and a large number of samples must be used, and the control of the film thickness is difficult, and therefore, the reproducibility is poor.

Furuno et al. Also show a method of forming an alignment film by accumulating a Langmuir-Blodgett film (LB film) of a purple film on an electrode, and detecting a photoelectric response as a current response. Although a method utilizing such a gas-liquid interface is also effective, it is difficult to produce a purple LB film in which the interface of proteins is denatured or the orientation is sufficiently controlled. In addition, sandwich cells made of LB films are made from the viewpoint of devices, but there is also a practical problem that very low current response of 10 ^-11 A is obtained even with several tens of accumulated films. .

For the protein orientation, several conventional methods have been tried. Fromherz et al. Attempted to adsorb proteins using the charge of lipids, utilizing the properties of lipid monolayers. Using a multi-chambered circular trough developed by them, they made an ammonium ion-doped LB film of methyl stearate, adsorbed ferritin, fixed it by glutaraldehyde treatment, and pulled it up onto the substrate (P. Fromherz) ; Nature231 2
67 ('71)).

When this was observed with an electron microscope, it was reported that the protein had a locally regular arrangement, but this was not a technique for arbitrarily orienting any protein.

On the other hand, E. Uqziris and R. Kornberg considered that binding of an antibody against hapten to a hapten phospholipid monolayer could cause protein orientation, and LB of dinitroferulphosphatidylethanolamine with dinitrophenyl group as papten. The film was prepared on an electron microscope grid on which carbon was deposited, and reacted with an anti-dinitrophenyl monoclonal antibody ("Nature" 301 125 ('83)).

When observed under an electron microscope, a two-dimensional crystal of the protein was observed as a hexagonal crystal pattern, indicating that the protein was oriented. However, there are few proteins other than antibodies that can bind to phospholipids, and this method is not universal.

(Problems to be Solved by the Invention) An object of the present invention is to firstly provide a color image light-receiving element having a high output reproducibility and a high response speed using an oriented thin film in which the front and back of a photosensitive dye protein are uniform. Second, it is to provide a color image light receiving element capable of controlling the film thickness and obtaining a stable response even with a very thin (several layers) cumulative film, and thirdly, to provide color sensitivity in the entire visible region. It is an object of the present invention to provide an element for receiving a full-color image signal by using a photosensitive protein thin film having the same.

(Means for Solving the Problems) An object of the present invention is to provide a hapten lipid and a photosensitive chromoprotein.
Antibodies (Bispec
ific antibody: BS antibody), which is a light-receiving element comprising a light-receiving unit obtained by supporting an alignment film formed by sequentially binding a hapten lipid, the antibody and a photosensitive dye protein on an electrode. The present invention has been attained by assembling a color image light receiving element having a plurality of combinations of light receiving units having photoelectric conversion functions having different photosensitive wavelength ranges.

More specifically, in the present invention, one antigen binding site recognizes a hapten, and the other one antigen binding site uses a BS antibody that recognizes either the cytoplasmic side or the extracellular side of a photochromic protein, Hapten on the matrix and this
An antigen binding site that recognizes the hapten of the BS antibody is bound,
Next, either the cytoplasmic side of the photosensitive chromoprotein or the outside of the cell is bound to an antigen-binding site of the BS antibody that recognizes the photosensitive chromoprotein.

In the present invention, a photosensitive chromoprotein which is a biological substance is used as the photoreceptor. These are biological proteins and their derivatives that absorb light and effectively convert the energy into chemical work.Therefore, the photochromic protein used in the present invention is not limited as long as it has such a function. And various photochromic proteins can be used, and the present invention is not limited to these types. Representative examples of the photochromic protein include, for example, visual pigments rhodopsin, bacteriorhodopsin, halorhodopsin, phoborodopsin,
Rhodopsin family such as arche-rhodopsin. Among these, the most preferred of the present invention is bacteriorhodopsin, which is excellent in stability in vitro.
Bacteriorhodopsin is a type of retinal protein that has opsin as a protein and retinal as a chromophore, similar to the visual pigment rhodopsin.
bacterium halobium), for example, D. Oest
erhalt, W. Stoeckenius, “Methods Enzymology” 31 , pp667
-678 (1974), and can be purified as a disc-shaped substance called purple membrane. This purple membrane is thought to consist of a structure in which the trimer of bacteriorhodopsin has a two-dimensional hexagonal lattice crystal structure, and the interstices are surrounded by boundary lipids (about 1/3 of the weight of rhodopsin) (R. Henderson and PNTUnwin, “Nature” 27
5 , pp 28-32 (1975)). Bacteriorhodopsin contains retinal (a vitamin A derivative) as a chromophore. Retinal has a Schiff bond with the ε-amino group of lysine, which is the 216th amino acid of the protein molecular chain, and a long wavelength shift called an opsin shift caused by this bond gives wide visible absorption.

In the method for orienting the photosensitive chromoprotein of the present invention,
An antibody having two antigen-binding sites in one molecule, one of which recognizes a hapten, and the other of which can recognize either the cytoplasmic side or the extracellular side of a photochromic protein is used. Such an antibody having two antigen-binding sites in one molecule is known as a BS antibody in fields such as missile therapy for cancer and immobilization of enzymes (M. Brennan et al.
See “Science” 229 81 (1985) and Murakami et al., “Proteins, Nucleic Acids and Enzymes” 33 217 (1988)). Antibodies are IgG, Ig
The antibodies are classified into M, IgA, IgD, and IgF classes, and any class of antibodies can be used for this purpose, but a particularly preferred class is IgG.

In the present invention, an LB membrane of a hapten phospholipid is preferably used as the hapten-supported matrix. Although various antigens can be used as the hapten, those having a dinitrophenyl group are particularly preferable.

In a preferred embodiment of the present invention, the outside of the bacteriorhodopsin (bRN terminal side) is oriented via a BS antibody on a phospholipid monolayer having a dinitrophenyl group (DNP) as a hapten.

The BS antibody used in this embodiment is obtained by binding an anti-DNP antibody to a bacteriorhodopsin N-terminal antibody (anti-bRN-terminal antibody).

The anti-DNP antibody conjugates 2,4-dinitrophenylsulfonic acid with limpet hemocyanin (KLH), immunizes the mouse and immunizes the mouse with mouse monoclonal anti-DN by a conventional method.
It can be prepared as a PIgG antibody.

The anti-bRN terminal antibody is composed of 9 residues of the N-terminal peptide of bacteriorhodopsin (△ Glu-Ala-Ihe-Thr-Gly-Arg-Pro-G
lu) can be prepared as a mouse monoclonal anti-bRN terminal antibody by solid phase synthesis using a peptide synthesizer and immunizing with a protein conjugated to KLH in the same manner as described above.

Against this monoclonal antibody, "Scienc
e "229,81 (1985), F (ab ') ₂ by digestion with pepsin, reduction with mercaptoethanolamine and protection of the mercapto group with sodium arsenite. And Ellman's reagent.

After treating the treated anti-DNP antibody with mercaptoethanolamine, the treated anti-bRN
Anti-DNP and anti-bRN end by reacting with terminal antibody
BS antibodies can be prepared.

In addition, there is a biological method called a hybrid hybridoma method developed by C. Milstein as a method for producing a BS antibody, and this method is also useful (“Immunol. Today” 5, 30).
0,1984).

An element for extracting the photoresponse of the photochromic protein as a physical signal is a hapten phospholipid on the electrode substrate.
A method in which an LB film is provided and the dye protein is oriented by applying the photosensitive dye protein orientation method of the present invention described above.

For example, a chloroform solution of dinitrophenylphosphatidylethanolamine is spread on a pure water phase to prepare a hapten phospholipid monolayer, and this monolayer is Sn
The O ₂ layer is transferred to a transparent electrode supported on a glass substrate, sufficiently incubated with the above-mentioned aqueous solution containing anti-DNP and anti-bRN terminal BS antibody, and then incubated with the purple membrane suspension isolated by the method of Oesterhelt Thereby, an element in which bacteriorhodopsin is oriented can be obtained.

As the light-receiving chromoprotein of the color image light-receiving element of the present invention, two or more kinds of photosensitive chromoproteins having different photosensitive wavelengths may be selected according to the desired light-receiving element configuration.

It is also possible to change the photosensitive wavelength range by changing the retinal portion of a natural protein. Its wavelength region and retinal derivative Tokunaga, Iwasa, "film" 9 73-91 are described in detail in ('84).

For example: 1.all-trans-retinal (maximum absorption 570 nm) 2.13-cis-retinal (maximum absorption 550 nm) 3.3,4-dihydroretinal (maximum absorption 593 nm) 4.5,6-dihydroretinal (maximum absorption 475 nm) 5. Retro- γ-retinal (absorption maximum at 430 nm). Furthermore, the bacteriorhodopsin gene has been cloned, and the nucleotide sequence of the DNA has been determined (RJ Dunn et al "Pro NAS" 78 6744-6
748 ('81)).

Based on these findings, bacteriorhodopsin derivatives having different absorption wavelength ranges can be obtained by changing a part of the amino acid sequence of the photosensitive chromoprotein using DNA recombination technology (HGKhorona et al. "J.Bio
l. Chim. " 262 9246-9254, 9255-9263, 9264-92, 70927
1-9276, 9277-9284 ('87) T. Mogi et al, "Por.
S. " 85 4148-4152 ('88)". The present invention can be achieved using such derivatives.

The photosensitive chromoprotein used in the present invention can be used by being mixed with various binder materials in the process of forming the thin film. Examples of the binder material include phospholipids, fatty acids, fatty acid esters, aliphatic amines, amphiphilic compounds such as aliphatic amides, collagen, albumin, cellulose, biopolymer compounds such as chickens, polyethylene oxide, polyvinyl alcohol, poly Synthetic polymer compounds such as acrylamide and polycarbonate are exemplified.

 Next, the color image light receiving element of the present invention will be described.

In the present invention, in a light receiving element for obtaining color image information as an electric signal, the element surface is composed of a plurality of electrodes that are electrically independent on a two-dimensional plane, and a photosensitive layer is formed on each of the electrodes. An alignment film of a sex chromoprotein is bonded. Each of these electrodes is called a light receiving unit (a so-called pixel). In the present invention, a light receiving element is formed by providing a plurality of combinations of two or more types of pixels having different photosensitive wavelengths.
There are the following two methods for combining two or more types of pixels having different photosensitive wavelengths.

A. A color image light-receiving element formed by combining two or more kinds of pixels having different photosensitive wavelengths and assembling them on the same plane to form one color pixel unit, and forming a plurality of such color pixel units on the same plane. This is the one described in FIG. 1 includes a blue pixel 1, a green pixel 2, and a red pixel 3 on one light receiving plane 5, respectively.
Are alternately arranged in a mosaic pattern, and each of the blue pixel 1, the green pixel 2,
The combination of the red pixels 3 constitutes one color pixel unit. Of course, the arrangement is not limited to such an arrangement, and a matrix or other arrangement is possible.

B. Pixels having the same photosensitive wavelength are arranged on the same plane to form a monochromatic image receiving plane, and this receiving plane is a group of pixels having different photosensitive wavelengths. A color image light receiving element formed differently from one or more. This is the one described in FIG. In the light receiving element of FIG. 2, a plurality of red pixels 3 are provided on a first light receiving plane 8, a plurality of green pixels 2 are provided on a second light receiving plane 7 thereon, and a third pixel 3 is provided thereon. A plurality of blue pixels 1 are provided on the light receiving plane 6.
In this case, a combination of two or more types of pixels having different photosensitive wavelengths (that is, a color pixel unit) is formed so as to overlap in the vertical direction as indicated by a broken line 10.

In A, the color image information is detected by one light receiving plane, while in B, the color image information is detected by a plurality of light receiving planes. In FIG. 2B, as shown in FIG. 2, the light as image information passes through the upper light receiving plane and then reaches the lower light receiving plane having a different color gamut. Therefore, in this case, the material constituting the light receiving plane except for the lowermost layer is preferably a light transmissive material including the electrode and its substrate. For the purpose of increasing the light transmittance, the layer of the photosensitive chromoprotein is also desirably a thin layer having a smaller optical absorption.

Comparing the above structures A and B, in A, the area occupied by three pixels gives one color pixel unit, whereas in B, the area occupied by one pixel is at least 1
It corresponds to a unit of color pixels. Therefore, in the present invention, it is preferable to use a multilayer structure of B in that the pixel unit is good and small.

Each individual pixel is independently connected to at least one conductor and connected to a circuit for information processing including a scan path including addressing of a light response signal. However,
When a photosensitive dye protein, a working electrode, and a counter electrode are used as at least three types of elements constituting a pixel, the counter electrode can be used as one common electrode for a plurality of light receiving units. FIG. 3 shows this configuration.
When a reference electrode is used as the third electrode in addition to these elements, it can be used as a common electrode.

The microelectrodes (mainly working electrodes) that make up the pixels are coated on the substrate using a vacuum deposition method, a sputtering method, or the like, and are coated with a photo resist method for pattern printing, a plasma method for etching, and an electrolytic method. By patterning the electrode shape using a wet etching method or the like, a fine pattern including electric wiring can be provided on the substrate.

In the present invention, as the electrode material capable of detecting an electric signal constituting a pixel which is a light receiving unit, various conductive metals (Au, Pt, Al, etc.) in addition to semiconductors including silicon, compound semiconductor, metal oxide semiconductor, and the like. Alternatively, a conductive metal oxide (SnO ₂ , In ₂ O ₃ , RuO ₂ , etc.) is preferably used. Among them, SnO ₂ , In ₂ O ₃ , and
And a thin film of these composites (ITO). Among these, SnO ₂ and ITO which are particularly preferably used in response in terms of the chemical stability of the electrode material and the S / N ratio in the photocurrent response in addition to the good light transmittance are SnO ₂ and ITO, which are SnO ₂ and ITO. The conductivity is preferably 10 ² Ω ^-1 cm ^-1 or more, more preferably 10 ³ Ω ^-1 cm ^-1 or more, as the conductivity.

These conductive electrode materials are supported as thin films on a transparent support such as glass or resin by a vacuum evaporation method or a sputtering method, and the thickness thereof is preferably 100 to 10,000 Å.
Particularly preferably, it is 500 to 6000 °.

Preferred structures of the element for photoelectric conversion used in the present invention include two types.

One of them is a photovoltaic pixel composed of a three-layered junction of an electrode / a photosensitive dye protein alignment film / electrode. In this type of cell, a relatively thick film (absorbance of 0.1 or more and thickness of 0.5 μm or more) is usually used as a photosensitive dye protein alignment film to obtain a sufficient electromotive force response.

The other is an electrochemical cell type pixel composed of a four-layer junction of electrode / photosensitive chromoprotein / ion conductive electrolyte / electrode. In this type of cell, an ultrathin film corresponding to several monolayers (several hundred degrees) of protein can be used for photoelectric conversion as a thin film of a photosensitive dye protein.

 The light response of this cell is detected as a photocurrent.

Of these two types of pixel structures, in the present invention, it is preferable to use the latter electrochemical cell type from the viewpoint that an ultrathin film can be used.

An electrochemical cell-type element basically comprises a conductive electrode substrate (working electrode), an oriented film of a photosensitive chromoprotein, an ion-conductive electrolyte, and at least three elements of a counter electrode. Are joined in this order. The device may include, in addition to these elements, a reference electrode, if necessary, as a third electrode element, the reference electrode being located in the ion-conducting electrolyte. The two or three types of electrodes may be connected to an external circuit, and a voltage may be externally applied between the working electrode and the counter electrode or the reference electrode.

In a configuration using three types of electrodes, one of useful as a setup of an external circuit including a current measuring device is
One is a potentiostat (potentiostat).

As the counter electrode, a material similar to the above-mentioned conductive electrode material is preferably used. However, when the element is a two-electrode system not including a reference electrode, the counter electrode desirably also has the performance as a reference electrode. Most preferably, a silver chloride electrode is used.

The electrolyte used as the ion-conductive medium in the electrochemical cell device includes an electrolytic aqueous solution and a solid electrolyte made of an inorganic or organic substance. Electrolyte solution is supported salt
It is an aqueous solution containing 0.01 M to 1 M, and as a supporting salt, for example, KC
l, NaCl, K ₂ SO ₄ , KNO _{3 and the} like are used. The pH of these aqueous solutions is preferably near neutral, but it is not preferable to include a buffer compound (buffer) for pH control. The pH is set using an acid or an alkali. It is preferable to use a solution that has been subjected to a deoxygenation treatment. As the solid electrolyte, for example, H ⁺ -WO ₃ system, Na ⁺ -β-Al ₂ O ₃ system, K
^In addition to inorganic compounds such as ⁺ -ZnO, PbCl / Kcl, and SnCl, salts are included as ion carriers in the medium of high molecular compounds such as gelatin, agar, polyvinyl alcohol, and general-purpose cation exchange resins and anion exchange resins. Can also be used.

In the case where an electrochemical cell type device is used as the photoelectric conversion device in the present invention, in order to provide a higher S / N ratio for the photocurrent response, the working electrode on which the photosensitive chromoprotein is carried is usually an electrochemical device. It is preferable to adopt a cathodic polarization state. The cathodic polarization state is
This is achieved by applying a negative bias to the working electrode from a counter electrode or a reference current from an external circuit. This bias is usually +1 with respect to the saturated calomel electrode (SCE).
−1, preferably −0.1 to −0.5V.

Examples of the present invention will be described below, but the means for detecting a color image in the present invention is not limited to these.

〔Example〕

(Preparation of bacteriorhodopsin) A purple membrane containing bacteriorhodopsin as a photosensitive pigment protein was isolated from Halobacterium halobium according to the method of Oesterhalt et al., And dispersed in pure water to prepare a dispersion having an absorbance of 14.0 (555 nm). Also, KSHuang et al., “Fed. Proc.” Vol. 40, p. 1659 (1
981), bacteriorhodopsin containing retro-γ-retinal having a maximum absorption wavelength of 430 nm was prepared according to the method described above, and this membrane fragment was dispersed in pure water to absorb light of 12.0 (430 nm).
Was prepared. In addition, F. Tokunaga and T. Ebrey,
Bacteriorhodopsin containing 3,4-dihydroretinal having a maximum absorption wavelength of 593 nm was prepared according to the method of "Biochemistry" Vol. 17, p. 1915 (1978), and this membrane was dispersed to obtain a dispersion having an absorbance of 12.0 (593 nm). Was prepared.

(Preparation of BS antibody) The antigen determination site is dinitrophenyl group by the following method.
A BS antibody molecule recognizing the bRN terminal 9 residues was prepared. First
Is the preparation of an antibody against the dinitrophenyl group and the 9 residues at the bRN terminal. First, a DNP-KLH conjugate was prepared with dinitrophenylbenzenesulfonic acid and KLH, and then the bRN-terminal peptide was bound to KLH using water-soluble DCC. This conjugate is used to transfer a group of BALB / C mice to DNP-K
Immunization was performed on the LH conjugate and the bRN terminal 9 residue peptide KLH conjugate.

After immunization, spleen cells of the immunized animal were prepared, and this was used to prepare MOPCs using the method described in Gulfre et al., (1981), Method in Enzymology, Vol. 73, pp. 3-46.
-21 was fused with myeloma cells (SP2 / 0-Ag14). Hybrid cells were selected in hypoxanthine-aminopterin-thymidine medium, cloned, and screened for the production of antibodies to the desired conjugate in the manner described above, such as Gulfre. Clones found to produce antibodies to the desired conjugate are then selected and
A clone producing an IgG-type antibody having a high affinity for the conjugate was selected. The clones of interest were stored in liquid nitrogen until use. Antibodies are
Cloned cells were prepared by growing in spinner flasks on Zurbecco's modified Eagle mordant medium containing 5% fetal calf serum. Alternatively, higher antibody yields can be obtained by a culture technique in which cells are grown as ascites tumors in the abdominal cavity of prestained rats.

Then, a desired IgG antibody against DNP and the bRN end was prepared,
Ai et al. (1978), Immunochemistry, Volume 15, Volume 42
Purified from ascites by affinity chromatography on Protein A-Sepharose as described on pages 9-436. Then, each of the two purified antibodies was
Hackett et al. (1981) Immunology, Vol. 4, No. 207
215pp. 215, converted to F (ab ′) ₂ fragment by treatment with pepsin as described below. 4 mg of purified immunoglobulin (IgG) is added to 0.1 M acid buffer (pH 4.6)
And cultured at 37 ° C. with 40 μg of pepsin. After 20 hours, the mixture is brought to pH 8.1 with Tris buffer.
And purified by gel filtration on Sephadex G-50, followed by passing through a column of Protein A-Sepharose.

The two F (ab ') ₂ fragments were then combined to generate a BS antibody as described below. First, one of the fragments was gently reduced with 10 mM mercaptoethylamine hydrochloride at 37 ° C. under a nitrogen atmosphere for 1 hour, and this fragment was treated with H
Separated into half molecules without breaking the bonds between the chains and the light chains. The mixture was then applied to a Dowex-50 column.
The reducing agent was removed by passing at pH5. The eluate was then immediately mixed with 0.02 M sodium phosphate (pH 8.0) and 3 mM EDTA as described in Laso and Griffin, Journal Immunology (1980), Vol. 125, pp. 2610-2616. 2,5'-dithiobis (2-nitrobenzoic acid)
And reacted. The Fab'-thionitrobenzoic acid derivative thus produced was then subjected to gel filtration on Sephadex G-100 to give 0.2M sodium phosphate (pH 8.0).
Was purified. The other F (ab ') ₂ fragment was similarly reduced and treated with Dowex-50 and the resulting Fa
The b 'derivative is immediately mixed with an equimolar amount of the Fab'-thionitrobenzoic acid derivative and incubated at 20 ° C for 3 hours to produce a mixture containing a high yield of BS antibody, where each determinant is a disulfide-bonded F (ab ') ₂ L-H condensed by
Consists of half molecules. This mixture is passed through a column of Sepharose 4B equilibrated with 0.1 M Tris (pH 7.5) to obtain a homogenous sample of the same BS antibody, which contains dinitrophenyl groups covalently attached thereto. The column is then washed with 0.1 M Tris (pH 7.5) and then the anti-D
The NP antibody was eluted with 0.1 M glycine (pH 2.5) and then neutralized with Tris.

The eluate was then covalently linked to bR by CNBr activation.
Passed through a second column of Sepharose 4B with N-terminal peptide. The column was washed with 0.1 M Tris (pH 7.5), and then the anti-DNP and anti-bRN terminal BS antibodies were added to 0.1 M glycine (pH 7.5).
Eluted in 2.5) and then neutralized with Tris. The desired BS antibody was obtained in the eluate.

(Electrode patterning) Film thickness 2000mm, conductivity 5 × 10 provided on glass substrate
An ITO thin film of ³ Ω ^-1 cm ^-1 was used as a working electrode, and the ITO thin film was divided into 1 mm ² electrically independent square electrodes having lead terminals with a width of 100 µm by patterning. Electrode patterning, naphthoquinone azides (photoresist)
Is applied on the electrode thin film, light irradiation is performed through the pattern image, a resist pattern is formed on the electrode through alkali treatment, then the electrode is treated with a Zn / HCl etching solution, and then the resist is dissolved in a solvent. The removal was carried out by the method of removing.

(Production of Element) Here, an example of producing a color light receiving element having a three-layer structure of blue, green, and red light receiving planes using an electrochemical cell type pixel will be described.

2000 mm thick transparent conductive electrode, conductivity 3 × 10 ³ Ω
A glass substrate carrying a fluorine-doped SnO ₃ film of ⁻¹ cm ⁻¹ was patterned as described above to form a large number of 1 mm ² square microelectrodes and lead terminals on the substrate. Further, a protective film (thickness: about 0.1 μm) made of a photocurable resist was placed on the lead wire only by patterning via a photomask, and the circuit portion was electrically shielded. Thus, a substrate having only the SnO ₂ working electrode corresponding to the light receiving unit exposed was manufactured.

Next, a chloroform solution (1 mM) of dinitrophenylphosphatidylcholine was developed on a pure water phase to prepare a monomolecular film at room temperature. The characteristics of surface pressure (Tc) -molecule occupation area (A) of the monomolecular film thus obtained were measured on a Langmuir film balance.

A dinitrophenyl phosphatidylcholine monolayer on the water surface is formed on the SnO ₂ surface of the patterned SnO ₂ substrate
The transfer operation was performed by the horizontal attachment method under a constant surface pressure of 300 dyn / cm. This thin film was dried by standing at room temperature for 1 hour, and incubated in the solution containing the BS antibody prepared in Example 1 for 1 hour. This was washed in pure water, incubated with the bacteriorhodopsin suspension prepared in the manner described above for 1 hour, and then washed in pure water for 1 hour.
Dried for hours. By the above operation, the hapten lipid (dinitrophenyl phosphatidylcholine molecule) is placed on the substrate of SnO ₂ as an electrode from the electrode surface side to the outside of the electrode,
BS with two types of antigen binding sites with different antigen specificities
The antibody and a bacteriorhodopsin molecule of a photosensitive chromoprotein carried an oriented film of a molecular complex in which three molecules were sequentially bonded and oriented.

Next, a hardened gelatin thin film (dry film thickness: 5 μm) impregnated with a 0.1 M KCl aqueous solution as a thin film of an ion-conductive polymer electrolyte was overlaid on the above thin film and brought into close contact with each other.

Further, a transparent glass electrode in which silver was deposited to an average thickness of 200 mm as a counter electrode and a reference electrode was superimposed on the gelatin electrolyte membrane so that the silver deposition surface was in close contact with the electrolyte membrane. In this way, a transparent thin-layer type green photosensitive light-receiving element comprising a junction of SnO ₂ / bacteriorhodopsin alignment film / electrolyte thin film (KCl) / AgCl-Ag was produced. This device showed extremely weak absorption (absorbance: about 0.005) derived from the purple film at a wavelength of around 560 nm. The element covered the cross section of the edge part with epoxy resin and shielded the electrolyte.

Then, using a blue-sensitive bacteriorhodopsin (absorption maximum of 430 nm) containing retro-γ-retinal as a dye prepared by the method described above, a blue-sensitive light-receiving element composed of pixels was obtained by the same process as that described above. Produced. In addition, a red-sensitive light-receiving element composed of pixels was produced using red-sensitive bacteriorhodopsin (absorption maximum 590 nm) containing 3,4-dihydroretinal as a dye in the same manner.

In each of the three thin-layer cells of the light-receiving elements having different photosensitive areas, a terminal independently taken from a working electrode of each pixel and a terminal taken from a common counter electrode (Ag) were connected to an external current measuring circuit. The current-side measurement circuit consists of a circuit in which a DC power supply and a current detection device are connected in series, and the electrochemical potential of the working electrode is controlled with respect to the Ag / AgCl electrode of the counter electrode also serving as the reference electrode. .

As shown in FIG. 2, the three types of light receiving elements were overlaid and fixed in the order of the blue light sensitive element and the green light sensitive element from the light incident side to construct a target color image light receiving element.

To measure the photocurrent response of these devices, 150 W was applied to the working electrode with a bias of -0.4 V applied to the counter electrode.
When a 550 nm band light was incident on the device from a xenon lamp through a color filter, a fast rising photocurrent response was observed in the pixels in the green photosensitive device. FIG. 6 shows the time change of this response.

The signal of the current measuring circuit was input to the plane-parallel image processing device, and the processed output signal was input to the color liquid crystal display device, thereby assembling a color image information display system. A 150 W xenon lamp is used to illuminate the three-color color light-receiving element through a color filter to illuminate three color characters consisting of blue, green, and red. These color characters are identified on the display element and displayed as an image. Was done.

[Brief description of the drawings]

FIG. 1 is a schematic view of the device of the present invention, wherein 1, 2 and 3 are blue light receiving units (pixels), green light receiving units and red light receiving units, 4 is color pixel units, and 5 is a light receiving plane including a substrate. Show. FIG. 2 is a schematic cross-sectional view of another example of the device of the present invention, wherein 1, 2, and 3 are the same as in FIG. 1, and 6, 7, and 8 are a blue light receiving plane, a green light receiving plane, and a red light receiving plane, respectively. And 9 indicate a support (substrate). FIG. 3 shows a color image receiving unit of the present invention comprising a laminated structure of working electrode / photosensitive chromoprotein / counter electrode; 1 is a transparent support; 2 is a working electrode; 3 is a counter electrode; 1 shows a blue-sensitive, green-sensitive, and red-sensitive photosensitive dye protein thin film. FIG. 4 shows an optical absorption spectrum of a thin film of a wavelength conversion type bacteriorhodopsin, 1 is a retro-γ-retinal bacteriorhodopsin, 2 is a normal retinal bacteriorhodopsin, and 3 is a 3,4-dihydroretinal bacteriorhodopsin. FIG. 5 is a circuit diagram connecting the device of the present invention and an image display device, wherein 1 is a counter electrode substrate, and 2 is a single pixel formed by a working electrode to which bacteriorhodopsin is bonded. V ₁ , V ₂ and V _n are signal conversion elements (elements such as FETs) belonging to each pixel P ₁ , P ₂ and P _n , 4 is a two-dimensional information parallel processing device for electric signals, 5 is 2 It is a color display device of a two-dimensional image. FIG. 6 is a graph showing the photocurrent response characteristics of the device of the present invention.

Claims (6)

(57) [Claims] An antibody (Bispecific) having antigen specificity for two antigens, hapten lipid and photochromic protein simultaneously.
(Antibody: BS antibody) a light-receiving element using a hapten lipid, an antibody and a photosensitive dye protein comprising a light-receiving unit obtained by carrying an alignment film that is sequentially bonded, A color image light receiving element comprising a plurality of combinations of light receiving units having photoelectric conversion functions having different photosensitive wavelength regions. 2. The color image light-receiving device according to claim 1, wherein said photosensitive chromoprotein is selected from bacteriorhodopsin and its analogs. 3. The color image light receiving element according to claim 2, wherein said BS antibody comprises at least an F (ab ') portion of a monoclonal IgG antibody. 4. The method according to claim 1, wherein one of the antibody-binding sites of the BS antibody binds to a hapten on a matrix, and the other binding site binds to either the cytoplasmic side or the extracellular side of the photochromic protein. The color image light receiving element according to claim 1. 5. The apparatus according to claim 1, wherein the light receiving unit having the photoelectric conversion function is constituted by an electrochemical cell having a structure in which a photosensitive chromoprotein and an ionic conductive electrolyte are joined. Color image light receiving element. 6. A color image light receiving element according to claim 1, wherein said electrode is made of tin oxide or indium oxide.